1. Field of the Invention
Fluorescent proteins and nucleic acids that encode fluorescent proteins are provided. Also provided are methods for their use, and reagents, devices and kits for use in these methods.
2. Description of the Related Art
Labeling of a protein, cell, or organism of interest plays a prominent role in many biochemical, molecular biological, and medical diagnostic applications. A variety of different labels have been developed and used in the art, including radiolabels, chromolabels, fluorescent labels, chemilluminescent labels, and the like, with varying properties and optimal uses. However, there is continued interest in the development of new labels. Of particular interest is the development of new protein labels, including fluorescent protein labels.
Green Fluorescent Protein (GFP) from the hydromedusa Aequorea aequorea/Aequorea victoria (A. victoria) was identified by Johnson et al., J. Cell Comp. Physiol. (1962) 60:85-104 as a secondary emitter of the jellyfish's bioluminescent system, transforming blue light from the photoprotein aequorin into green light. The cDNA encoding A. victoria GFP (avGFP) was cloned as reported in Prasher et al., Gene (1992) 111:229-33 (SEQ ID NO:24). When ectopically expressed, this gene will produce a fluorescent protein due to its unique ability to independently form a chromophore (Chalfie et al., Gene (1992) 111:229-233). This finding has enabled broad applications for the use of GFP in cell biology as a genetically encoded fluorescent label.
Genes encoding fluorescent proteins have since been cloned from organisms of a wide variety of different phylogenetic clades including, but not limited to: Hydrozoa, Anthozoa, Arthropoda (Copepoda) and Chordrata (Brachiostoma), e.g., as reported in: Matz et al., Nat. Biotechnol. (1999) 17: 969-973; Chudakov et al., Trends Biotechnol. (2005) 23: 605-613; Shagin et al., Mol. Biol. Evol. (2004) 21: 841-850; Masuda et al., Gene (2006) 372: 18-25; Deheyn et al., Biol. Bull. (2007) 213: 95-100; and Baumann et al., Biol. Direct. (2008) 3: 28. Currently, the fluorescent protein (FP) family (also referred to in the art as the “GFP family”) includes hundreds of member proteins. While these proteins may collectively be referred to as members of the “GFP family”, emission maxima may vary widely in terms of wavelength, and therefore not all members of the family fluoresce green.
Proteins of the GFP family share a common GFP-like domain. This domain can be easily identified in the amino acid sequences of the various family members using available software for the analysis of protein domain organization, e.g., by using the Conserved Domain Database (CDD) program available at the website formed by placed “http://www.” in front of “ncbi.nlm.nih.gov/Structure/cdd/” and the Simple Modular Architecture Research Tool (SMART) program available at the website formed by placing “http://smart.” in front of “embl-heidelberg.de/”. For example, the GFP-like domain of avGFP begins at amino acid residue 6 and ends at amino acid residue 229. It has been demonstrated that a core domain within this domain, the “minimum GFP-like domain,” produced by truncating the protein at the N-terminus (up to 9 amino acid residues) and C-terminus (up to 11 amino acid residues) is sufficient to provide for maturation and fluorescence of GFP family proteins (Shimozono et al., Biochemistry. 2006; 45(20): 6267-71). Thus, when expressed, both GFP-like domain polypeptides and minimum GFP-like domain polypeptides can produce a protein that exhibits fluorescence.
The GFP-like domain comprises a chromophore that is responsible for the fluorescence emitted by fluorescent proteins upon irradiation with excitation light at an appropriate wavelength. The chromophore is formed by amino acids corresponding to the Ser65-Tyr66-Gly67 region of avGFP. Corresponding amino acids in fluorescent proteins other than avGFP can be determined by aligning the amino acid sequence of a protein under examination with avGFP (SEQ ID NO:24), e.g., as described in Matz et al., Nat. Biotechnol. (1999) 17: 969-973. As used herein the term “fluorescent protein” or “fluoroprotein” means a protein that is fluorescent; e.g., it may exhibit low, medium or intense fluorescence upon irradiation with light of the appropriate excitation wavelength. The fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues that act by themselves as intrinsic fluors, i.e., tryptophan, tyrosine and phenylalanine. As used herein, the term “fluorescent protein” also does not include luciferases, such as Renilla luciferase.
In fluorescent proteins of the GFP family, the chromophore is formed autocatalytically, i.e. no enzymes, cofactors and/or substrates are required for chromophore formation and fluorescence with the exception of molecular oxygen. It has been demonstrated that the green chromophore in GFP is formed by cyclization of the protein backbone in the Ser65-Tyr66-Gly67 region, followed by dehydrogenation of the Cα-Cβ bond of Tyr66. As a result, a bicyclic structure of 5-(4-hydroxybenzylidene)-3,5-dihydro-4H-imidazol-4-one is formed, in which the six-member aromatic ring of the Tyr66 side chain is linked to an unusual five-member heterocycle, which itself originates from condensation of the carbonyl carbon of Ser65 with the nitrogen of Gly67 (see e.g., Heim et al., Proc Nat'l Acad. Sci. USA. (1994) 91:12501-12504; Ormo et al., Science (1996) 273:1392-1395; and Yang et al., Nat. Biotechnol. (1996) 14:1246-1251). All of the green proteins possess the avGFP-like chromophore, with modifications of protein's environment contributing to differences in the spectral shapes of these different proteins (see e.g., Brejc et al., Proc. Nat'l Acad. Sci. USA (1997) 94: 2306-2311; Palm et al., Nat. Struct. Biol. (1997) 4:361-365; and Gurskaya et al., BMC Biochem. (2001) 2:6).
In red GFP-like proteins, additional chemical modification of the GFP-like chromophore occurs. In particular, oxidation of a Cα-N bond at residue 65 (avGFP numbering) results in an acylimine group conjugated to a GFP-like core in DsRed (see Gross et al., Proc. Nat'l Acad. Sci. USA (2000) 97:11990-11995; Wall et al., Nat. Struct. Biol. (2000) 7:1133-1138; and Yarbrough et al., Proc. Nat'l Acad. Sci. USA (2001) 98:462-467). The DsRed-like chromophore is formed within many other proteins with red-shifted absorption and fluorescence (See e.g., Pakhomov, A. A. and Martynov, V. I., Chem. Biol. (2008) 15: 755-764). In some proteins, the acylimine moiety of the DsRed chromophore is further attacked by various nucleophiles to form additional types of red-shifted chromophores. For example, the chromophore in the purple chromoprotein asFP595 is formed by hydrolysis of the acylimine group, resulting in cleavage of the protein backbone and formation of a keto group conjugated to a GFP-like chromophore core (see e.g., Quillin et al., Biochemistry (2005) 44: 5774-5787; and Yampolsky et al., Biochemistry (2005) 44: 5788-5793). In the orange fluorescent proteins mOrange and mKO, nucleophilic addition of Thr65 (in mOrange) or Cys65 (in mKO) side chain groups leads to unusual heterocycles without protein backbone scission (see e.g., Shu et al., Biochemistry (2006) 45: 9639-9647 and Kikuchi et al., Biochemistry (2008) 47: 11573-11580).
Amino acid substitution of one or more residues in the chromophore and chromophore environment will strongly affect fluorescence maxima of FPs. These positions crucial for fluorescence of particular color can be found by sequence comparison of fluorescent proteins of different colors. In many cases, one amino acid substitution, i.e. corresponding to residue 65 of avGFP, is required to produce a green fluorescent protein from the red FP (see e.g., Gurskaya et al., BMC Biochemistry (2001) 2:6).
Three-dimensional structure of the GFP-like domain represents so-called β-can, a 11-stranded β-barrel enclosing an α-helix (see e.g., Ormo et al., Science (1996) 273: 1392-1395; Wall et al., Nat. Struct. Biol. (2000), 7: 1133-1138; Yarbrough et al., Proc. Nat'l Acad. Sci. USA (2001) 98: 462-467; Prescott et al., Structure (Camb) (2003) 11: 275-284; Petersen et al., J. Biol. Chem. (2003) 278: 44626-44631; Wilmann et al., J. Biol Chem (2005), 280: 2401-2404; Remington et al., Biochemistry (2005) 44: 202-212; and Quillin et al., Biochemistry (2005) 44: 5774-5787). The chromophore is located in the central region of the α-helix.
Fluorescent proteins are widely known today due to their use as fluorescent markers in biomedical sciences (see, e.g., detailed discussions in Lippincott-Schwartz and Patterson in Science (2003; 300(5616):87-91) and Stepanenko et al. in Curr Protein Pept Sci. (2008; 9(4):338-369)). They are applied for wide range of applications including the study of gene expression and protein localization (Chalfie et al., Science 263 (1994), 802-805, and Heim et al. in Proc. Nat. Acad. Sci. (1994), 91: 12501-12504), as a tool for visualizing subcellular organelles in cells (Rizzuto et al., Curr. Biology (1995), 5: 635-642), and for the visualization of protein localization and transport along the secretory pathway (Kaether and Gerdes, FEBS Letters (1995), 369: 267-271), etc.
For fluorescent proteins suitable for such uses, novel fluorescent proteins have been identified with improved fluorescence intensity and maturation rates at physiological temperatures, modified excitation and emission spectra, and reduce oligomerization and aggregation properties. In addition, mutagenesis of known proteins has been undertaken to improve their chemical properties. Finally, codon usage may be optimized for high expression in the desired heterological system, for example in mammalian cells (Haas, et al., Current Biology (1996), 6: 315-324; Yang, et al., Nucleic Acids Research (1996), 24: 4592-4593).
For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths (Shcherbo et al., 2007; Shcherbo et al., 2009; Hoffman, Trends Biotechnol. 2008, 26(1): 1-4; Deliolanis et al., J Biomed Opt. 2008, 13(4): 044008).
Red and far-red fluorescent proteins are also important tools for multicolor labeling techniques (Chudakov et al., Trends Biotechnol. 2005; 23(12):605-613), enhanced FRET (fluorescent resonance energy transfer) techniques (Chudakov et al., Trends Biotechnol. 2005; 23(12):605-613) and visualization in living tissues (Shcherbo et al., Nat. Methods. 2007; 4(9): 741-746; Shcherbo et al., Biochem J. 2009; 418(3): 567-74; Hoffman, Trends Biotechnol. 2008, 26(1): 1-4; Deliolanis et al., J Biomed Opt. 2008, 13(4): 044008).
Katushka (also known as TurboFP635, SEQ ID Nos: 2) is one of the brightness far-red fluorescent proteins known in the art. Katushka was produced from the red fluorescent protein from sea anemone Entacmaea quadricolor (Shcherbo et al., Nat Methods. 2007; 4 (9):741-6). It is dimeric protein characterized by excitation maximum at 588 nm, emission maximum at 635 nm, quantum yield 0.34, extinction coefficient 65,000 M−1 cm−1 (calculated brightness 22.1), and pKa 5.5.
The utility of far-red fluorescent proteins as a tool in molecular biology has prompted the search for such proteins with improved properties, as compared to known far-red fluorescent proteins. Thus, it is an object to provide novel fluorescent proteins that exhibit properties not currently available in the known fluorescent proteins, as well as DNAs encoding them.